1. Field of the Invention
This invention pertains to non-covalent-carrier-hapten bioconjugates and their use to selectively deliver diagnostic and therapeutic agents to target tissues and organs. Particularly, this invention pertains to the selective delivery of fluorescent non-covalent carrier-hapten bioconjugates to a specific site for the purpose of determining the structure and function of tissues or organs using optical tomographic imaging procedures, fluorescence monitoring procedures, absorbance monitoring procedures, or endoscopic examination procedures.
2. Description of the Prior Art
Non-covalent intermolecular forces (e.g. electrostatic, hydrogen bonding and Van der Waals interactions) play a vital role in many biological process such as enzyme catalysis, drug-receptor interaction, antigen-antibody interaction, biotin-avidin interaction, DNA double helix formation, phagocytosis, pigmentation in plant and animals, and cellular transport. Variety of colors observed in flowers and plants, for instance, are attributed to non-covalent association between the natural pigments and carbohydrates or proteins found in plant cells.
Non-covalent forces can alter the physicochemical and/or biological properties of haptens or carriers. For example, association of dye or a pigment molecule with proteins or carbohydrates can change the chemical or photo stability, change the intensity and/or the wavelength of absorption/emission maxima, or both. Although the interaction energy per unit interaction is quite small (c.a. less than 40 kJ/interaction), the cumulative effect of multiple points of interaction along the two surfaces can be substantial and can lead to strong binding between the hapten and the carrier. This approach has been successfully used to prepare anti-DNA antibodies. DNA is a highly charged anionic macromolecule that is normally non-immunogenic; but, when it is complexed with a highly charged cationic methylated bovine serum albumin (MBSA), DNA becomes immunogenic. Thus, the non-covalent DNA-MBSA bioconjugate was stable enough to elicit immune response toward DNA.
Non-specific interactions also play an important role in biological processes. For example, human serum albumin binds various molecules in a non selective fashion and facilitates the transport of these molecules across from the vasculature to the cells.
Thus, it is clear from the examples above that the non-covalently attached bioconjugates function biologically as a single unit. In addition to the above mentioned properties, the carrier molecule may also protect some haptens from chemical, photochemical, or radiolytic degradation. The present invention is intended to exploit the concept of non-covalent interactions in the design of novel bioconjugates for diagnosis and therapy.
Further, the use of fluorescent dyes and covalent dye bioconjugates for the detection of various species in the body fluids is well known in the field of in-vitro immunodiagnostics. Dye-immunoconjugates have been widely used in immunohistology, and immunochemical detection of various antigens, hormones, drugs and the like in body fluids via radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) procedures. Fluorescent dyes have also been used as chemosensors and in fluorescence microscopy. (J. P. Desvergne and A. W. Czarnik, Chemosensors of Ion and Molecule Recognition, Kluver Academic, Boston, 1997; B. H. Satir, Ed., Noninvasive Techniques in Cell Biology, Wiley-Liss, New York, 1990). Indocyanine green, a highly fluorescent polyene dye, has been used for monitoring cardiac output, assessing hepatic function, and tomographic imaging of tumors (K. Licha et al., Synthesis and Characterization of Cyanine Dyes as Contrast Agents for Near-Infrared Imaging, SPIE, 1996, Vol. 2927, 192-197; X. Li et al., Tumor Localization Using Fluorescence of Indocyanine Green in Rat Models, SPIE, 1995, Vol. 2389, 789-798; B. Riefkce et al., In Vivo Characterization of Cyanine Dyes as Contrast Agents for Near-Infrared Imaging, SPIE, 1996, Vol.2927, 199-208).
In conventional bioconjugate chemistry, conjugates are prepared by covalent attachment of various effector molecules such as drugs, hormones, radiopharmaceutical agents, magnetic resonance imaging agents, chemotherapeutic agents, and the like to the bioactive carriers. Such a process often involves cumbersome chemical manipulation of the two components, in addition to the complicated synthesis of appropriate activated haptens necessary for covalent attachment. Moreover, the bioactivity of the resulting complex is, in many cases, either greatly diminished or obviated altogether.
Polyene dyes, in general, and indocyanine green, in particular, have several disadvantages that limit their use as near-infrared (NIR) contrast agents. Indocyanine green has a very short plasma half-life and is rapidly taken up by the liver (D. K Meijer et al., Pharmacokinetics of Biliary Excretion in Man. VI. Indocyanine Green, Eur. J Clin. Pharmacol., 1988, Vol. 35, 295-303); has low fluorescence efficiency, and undergoes degradation in aqueous media with loss of fluorescence. Therefore, there is a need for novel bioconjugates that are simple to prepare and stable enough to be useful as a diagnostic or a therapeutic agent.
It is, therefore, an object of the present invention to provide non-covalent carrier-hapten bioconjugates that are simple to prepare and are useful in diagnostic and therapeutic medical procedures.
It is another object of the present invention to provide bioconjugates that are stable during storage before being used in diagnostic and therapeutic medical procedures.
It is a further object of the present invention to provide a method for performing a diagnostic or therapeutic procedure on a patient.
It is a another object of the present invention to provide a method for altering the blood persistence of a hapten.
It is a another object of the present invention to provide a method for increasing the in vitro and in vivo fluorescent life of a fluorescent dye.
These and other objects are achieved using new and structurally diverse non-covalent carrier-hapten bioconjugates having the formula:
HM-CM
wherein HM is a hapten molecule whose molecular weight is generally, but not always, less than 1000 Daltons and is capable of performing specific functions; CM is a carrier molecule, whose molecular weight is generally, but not always, more than 1000 Daltons and is capable of transporting the hapten to a specific site; and the dashed line is a non-covalent bond between the carrier molecule and the hapten molecule.
Preferably, the bioconjugates are formed from fluorescent dye haptens and carrier molecules selected from the group consisting of serum albumin, methylated serum albumin, polypeptides with molecular weight ranges from 2000 to 20000 Daltons, and polysaccharides with molecular weight ranges from 2000 to 20000 Daltons, polynucleotides with molecular weight ranges from 2000 to 100000 Daltons, cyclodextrins, calixarenes, and surfactants.
Most preferably, the bioconjugates are formed from fluorescent dye haptens selected from the group consisting of cyanine, indocyanine, squaraine, porphyrrins, Rose Bengal, and methylene blue dye and carrier molecules selected from the group consisting of methylated serum albumin, polyarginine, polyaspartic acid, polyglutamic acid, cyclodextrin, inulin, polyadenylic acid, and polyguanylic acid.
The bioconjugates are useful as diagnostic and therapeutic agents in medical procedures because they are stable in vitro before being used and stable in vivo during and after use. It is theorized that the bond between the hapten and the carrier stabilizes the bioconjugates and delays its degradation in vitro by environmental factors. Also, the bond causes the bioconjugates to be metabolized slowly in vivo and therefore retained in the body for a longer period than expected for the non-bound hapten.
Other objects, advantages, and novel features of the present invention will become apparent in the following detailed description of the invention.